Project Summary Osteoarthritis (OA) is associated with severe joint pain, inflammation, and chronic cartilage degeneration. Mesenchymal stem cells (MSCs) derived exosomes are emerging as promising therapeutics for OA as they carry proteins and genetic materials that induce regenerative processes like cell migration, proliferation, differentiation and matrix synthesis. Their role in biological and transport crosstalk across multiple joint tissues and cell types, however, remains unclear. Additionally, the negative charge of exosome lipid bilayer hinders their penetration into the negatively charged cartilage. The high negative fixed charge density of cartilage offers a unique opportunity to utilize electrostatic interactions to enhance intra-tissue transport, uptake, and retention of exosomes by making them positively charged. We have designed an amphipathic cartilage penetrating cationic peptide (CP) that can rapidly diffuse through full tissue thickness due to their optimal charge, be up-taken by cells, and bind within for extended periods in both healthy and arthritic cartilage. This project will engineer cartilage targeting MSC-exosomes anchored with CPs and with an anti-catabolic OA biologic, IL-1Ra (IL-1 inhibitor) in optimal concentrations. Currently, extensive genetic engineering approaches are used to produce customized exosomes encapsulating biologics, which may compromise their intrinsic composition making their clinical translation complex. The project will use a simple one-step synthesis of grafting CP and IL-1Ra on exosome lipid bilayer. CP-exosomes can thus use cartilage as a drug depot and target cells thereby enhancing the availability of optimally loaded IL-1Ra to its receptors while preserving their intrinsic therapeutic potential. Aim 1 will engineer CP grafted MSC-exosome (CP-Exo) and characterize its intra-cartilage transport properties in healthy and arthritic states. Their transport crosstalk and uptake across multiple cell types using cytokine challenged chondrocyte and synovial cell co-cultures will be studied to understand whether their therapeutic benefits arise from cartilage or synovium targeting or both. Aim 2 will synthesize recombinant lipid fused IL-1Ra that will be anchored in different densities on exosome bilayer to form a hybrid vehicle, IL-1Ra-CP-Exo. Its bioactivity will be evaluated using cytokine challenged cartilage-synovium explant co-cultures and compared with free IL-1Ra and unmodified exosomes. Aim 3 will characterize joint kinetics, intra-cartilage uptake and biodistribution of CP-Exo in healthy and injured rat knees, and bio efficacy of IL1-Ra-CP-Exo in suppressing injury induced catabolic signaling will be evaluated using rat models of post traumatic OA. The project paves way for utilizing the intrinsic therapeutic potential of exosomes for cartilage repair as well as for its customizable development as a drug carrier allowing for adjustable intra-cartilage transport properties, easy drug anchoring and controllable loading of a variety of pro-chondrogenic protein drugs and antibodies. The success of this project can enable rapid clinical translation of exosomes as a cell-free, non-immunogenic platform for drug delivery to cartilage and other negatively charged tissues like meniscus, intervertebral discs, eye etc.